Field of Invention
The present invention relates to methods and systems for single molecule based nucleic acid amplification and subsequent detection of nucleic acid molecules, and particularly to the determination of SNPs, mutations, and to the diagnosis of diseases associated with the changes of these nucleic acid molecules.
Description of Related Art
Polymerase chain reaction (PCR) has been widely used in many areas of nucleic acid analysis for decades. Single molecule based PCR, also known as digital PCR, is a relatively new development of PCR technology. In single molecule PCR, the sample is diluted and divided into many individual nucleic acid amplification reactions, with less than one copy of template on average for each reaction. Some of the reactions have no template molecule, some have more than one copy of template molecules, and a certain percentage of the reactions have just one copy of the template. All the reactions are carried out in parallel. The nucleic acid amplicons in these reactions that start with just one copy of template molecule can be detected with methods known in the art. The analytical results provide either “yes” or “no” binary signals, as if in a digital format 0 or 1, with regard to whether a particular target molecule of interest is in the sample. Results from all the reactions in a digital PCR are statistically analyzed for quantitation of the target molecule. Digital PCR transforms the exponential amplification of conventional PCR into a linear relationship, and converts traditional analogue signals into a digital format. More detailed description of digital PCR can be found in Vogelstein et al. Proc. Natl. Acad. Sci. USA, 1999, 96, pp. 9236-9241, and U.S. Pat. No. 6,143,496, all herein are incorporated by reference.
The application of digital PCR includes mutation detection for early cancer diagnosis, assessing allele imbalance, prenatal genetic testing, quantification of gene expression, and DNA methylation status analysis. Pohl et al. Expert Rev. Mol. Diagn, 2004, 4(1):41-47; Blow, Nature Methods, 2007, 4(10):869-875; Diel et al. Curr Opin Oncol, 2007, 19:36-42; Dressman et al., PNAS, 2003, 100(15):8817-8822; Weisenberger et al. Nucleic Acids Res., 2008, 36(14):4689-4698; all are herein incorporated by reference. The single molecule amplification principle that digital PCR is based on is also used in the current next-generation DNA sequencing technologies, such as Roche Life Sciences' DNA pyrosequencing technology, Life Technologies' SOLiD DNA sequencing technology, and Illumina's DNA sequencing-by-synthesis technology, for sequencing template preparation.
One of the advantages of digital PCR is its capability of detecting and quantifying rare sequence events, such as mutation, in a large background of related template molecules. This is because each amplification reaction can be independent of other target molecules in the sample due to the fact that template molecules are divided into individual PCR reactions by limiting dilution. Such capability allows for the non-invasive early cancer detection from body fluid, such as plasma and stool samples from colorectal cancer patients, as described in Diehl et al. PNAS, 2005, 102(45):16368-16373; Diehl et al. Gastroenterol., 2008, 135(2):489-498; all are herein incorporated by reference. This technology also enables non-invasive prenatal genetic testing in the presence of maternal DNA, as described in Lo et al., PNAS, 2007, 104(32):13116-13121, which is herein incorporated by reference.
The quantitation with digital PCR is carried out by counting discrete positive PCR reactions in the sample. The differentiation between mutant and wild-type DNA molecules is achieved by determining the identities of the resulting amplicons. The ratio of mutant to wild-type DNA molecules is statistically calculated from these results. Digital PCR can also be used for absolute quantitation of target molecules. More details of digital PCR quantitation can be found in: Dube et al., PLoS ONE, 2008, 3(8), e2876; Warren et al. “The Digital Array Response Curve”, unpublished, Stanford University website thebigone.stanford.edu/quake/publications/DigResCurve.pdf; all are herein incorporated by reference. The precision and accuracy of digital PCR could be improved by increasing the number of target molecules being analyzed, i.e. more PCR reactions being screened in parallel for the measurement.
Currently, digital PCR is performed in a 384 well plate or a 48x770 Digital Array Nanofluidic Biochip from Fluidigm Corporation (South San Francisco, Calif., US). The majority of these applications utilize real-time PCR to analyze the amplicons in each reaction. There are reports of a method named BEAMing that generates nucleic acid amplicons on magnetic beads by emulsion PCR and uses flow cytometry to detection and counts those beads with amplicons. However, these digital PCR methods have limited multiplex capability because of the limitation of optical resolution of available fluorescent dyes.
Other means of generating nucleic acid amplicons from a single molecule include rolling circle amplification (RCA). RCA is an isothermal amplification method in which a nucleic acid probe is either hybridized or ligated onto a target nucleic acid molecule whose sequence is subsequently duplicated many times using a primer that is complimentary to part of the probe sequence. More detailed description of RCA and its applications can be found in Eriksson et al., J. Microbiol. Methods, 2009, 78, 195-202; Baner et al., Nucl. Acids Res., 1998, 26(22):5073-5078; Baner et al., Curr. Opinion Biotech., 2001, 12, 11-15; all are herein incorporated by reference. RCA is also used in sample preparation for the next generation of DNA sequencing, Drmanac et al., Science, 2010, 327:78-81, which is herein incorporated by reference.